Coding

Part:BBa_K3505023

Designed by: Asteria Tsapadikou,Emmanouil Stylianakis   Group: iGEM20_Thessaly   (2020-10-20)
Revision as of 19:31, 27 October 2020 by Asteriat (Talk | contribs) (→‎References)


FFAR2:V2tail:TCS GB compatible with B2-B3



Usage and Biology

FFAR2 is a naturally found eukaryotic GPCR protein that exhibits high affinity for SCFAs (acetic, propionic and butyric acid)(Kaemmerer, 2010) . After background searching through the NCBI database, we have identified the gene coding sequences needed for the designing of this gene construct. More specifically, a vasopressin receptor 2 segment has been attached to the C-terminal of the receptor, as it mediates recruitment of a TEV tagged b-arrestin-2, along with a TEV cleavage site(TCS).

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is presents in pUPD2 and has overhangs compatible for Golden Braid cloning. The CDS has position B2-B5.

Figure 1.The overhangs of this part in the Golden Braid Grammar.
Figure 2. The level 0 module : pupd2-FFAR2:V2tail:TCS (illustration from SnapGene)

Verification of Cloning

Fig.1:(U=Uncut C=Cut) Restriction Digestion of FFAR2:V2tail:TCS with BamHI, Expected bands : 3204 bp, Positive Result:C2,C3

Source

Synthesized by IDT.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 284
    Illegal PstI site found at 864
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 284
    Illegal PstI site found at 864
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 106
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 284
    Illegal PstI site found at 864
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 284
    Illegal PstI site found at 864
    Illegal NgoMIV site found at 364
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 560
    Illegal SapI site found at 815
    Illegal SapI.rc site found at 17


References

  • Kaemmerer, E., 2010. Fatty acid binding receptors in intestinal physiology and pathophysiology. World Journal of Gastrointestinal Pathophysiology, 1(5), p.147.

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